PROJECT SUMMARY Glioblastoma (GBM) is the most malignant brain cancer, with extremely poor prognosis in patients. Distinct molecular subtypes, characterized as proneural (PN), classical, and mesenchymal (MES), as well as inherited heterogeneity render GBM tumors resistant to current therapies. In addition to the genetic heterogeneity arising from the differentiated cells, GBM tumors also contain a small population of glioma-initiating or stem-like cancer cells (GSCs). Gene expression profiling studies from our lab showed that patient-derived GSCs can also be classified into subtypes phenotypically similar to GBM. We found over three thousand genes that are differentially expressed between PN and MES-like GSCs. Among these, Lymphocyte Antigen Complex 6, Locus K (LY6K) was one of the top differentially expressed genes. LY6K is a GPI-anchored protein from the LY6 family. Various members of the LY6 family have been implicated in human cancers, including breast, esophageal, and lung cancers. In our preliminary studies, we tested the roles of LY6K upregulation in GBM tumorigenesis and started to examine the underlying mechanism of LY6K action. We found that 1) high levels of LY6K expression correlates with poor prognosis of patients with GBM; 2) suppression of LY6K expression in MES-like GSCs significantly reduced tumorigenic behaviors in vitro and in vivo; 3) LY6K and EGFR-ERK signaling both become activated in irradiated PN-like GSCs which otherwise have undetectable levels of both; 4) suppression of LY6K expression decreases ERK1/2 activation, which is likely a downstream effect of EGFR signaling. Thus, we hypothesize that high levels of LY6K in GSCs promotes GBM tumorigenesis. Moreover, based our radiation data, we hypothesize that LY6K may function by enhancing EGFR signaling and subsequent ERK activation, thus promoting radioresistance. To test these hypotheses, we have developed two specific aims. The first aim examines the function of LY6K as an oncogene in GSCs. We will perform in vitro and in vivo tumorigenicity assays to determine whether modulation of LY6K expression in GSCs regulates tumorigenicity. We will also examine the mechanism of LY6K expression in GSCs and GBM clinical samples by examining LY6K promoter methylation. Secondly, we will investigate the relationship between LY6K and EGFR signaling, as it pertains to radioresistance. To determine whether LY6K modulates EGFR signaling, we propose to modify the expression of LY6K in GSCs and measure the resulting changes in pERK. Subsequently, we will determine which specific domains within LY6K are responsible for EGFR signal enhancement, via domain-deletion experiments. The insight gained from these aims will further our understanding the function LY6K in EGFR signaling as well as GBM tumorigenicity.